The present invention is in the general field of detection of biological molecules, including DNA, RNA, protein and the like, and specifically in the field of detection of RNA:DNA hybrids on a solid phase, as further described herein, using a hybridization assay.
The RNA or DNA for many genes, including those associated with disease states, and microorganisms and viruses have been isolated and sequenced. Nucleic acid probes based on such sequences are currently available to identify a large number of genes and infections. Nucleic acid probes are detectable nucleic acid sequences that hybridize to complementary RNA or DNA sequences in a test sample. Detection of the probe indicates the presence of a particular nucleic acid sequence in the test sample for which the probe is specific. In addition to aiding scientific research, nucleic acid probes may be used to detect the presence of viruses and microorganisms such as bacteria, yeast and protozoa as well as genetic mutations linked to specific disorders in patient samples.
Grunstein, et al, Proc. Natl. Acad. Sci. USA 72:3961 (1975) and Southern, J. Mol. Biol. 98:503 (1975) describe hybridization techniques using radiolabeled nucleic acid probes. Nucleic acid hybridization probes have the advantages of high sensitivity and specificity over other detection methods and do not require a viable organism. Hybridization probes are often labeled with a radioactive substance that may be easily detected.
The existing hybridization techniques that utilize radioisotopes to label probes introduce additional expenses caused by the high costs of disposal of radioactive waste products and the need for monitoring personnel and the workplace for contamination. In addition, the short half-life of radioactive compounds such as 32p requires that radioactive probes be produced frequently. Radioactive nucleic acid hybridization is therefore discouraged in commercial areas such as clinical diagnosis.
Probes have been indirectly labeled in an attempt to avoid the problems associated with direct radioactive labeling. One common method of indirect labeling is to attach biotin, a small vitamin, to the nucleic acid probe using a chemical or enzyme technique. Following hybridization to the specific nucleic acid, the biotin is detected by reaction with streptavidin, a protein that binds biotin tightly and has been labeled with an enzyme or fluorochrome. Bound biotin-streptavidin complex may be detected by reaction with color-producing substrates and the fluorochrome may be seen when reacted with incident light of appropriate wavelength. However, indirect labeling of hybridization probes with biotin or other haptens often increases the xe2x80x9chydrophobicityxe2x80x9d of the probe. The probe tends to interact non-specifically with materials other than the complementary nucleic acid target, leading to high background. The biotin label increases non-specific binding, which leads to high background, thereby reducing sensitivity and increasing the likelihood of a false-positive result. Indirect labeling is also less sensitive than direct labeling because the labeling density is limited; only a small fraction of the bases are labeled giving a limiting number of sites for signal generation. An increase in the labeling density of a probe leads to increased non-specific binding, higher background, and ultimately, failure of the probe to hybridize with its target due to the interference of the hapten with base pairing. Indirectly labeled probes are therefore not well suited to clinical diagnosis because of its inaccuracy and false positive results.
Hybridization of a probe to the specific nucleic acid sequences has been detected with the use of an intercalating agent such as acridine orange or ethidium bromide as described in U.S. Pat. No. 4,563,417 to Albarella et al. The intercalating agent becomes inserted between hybridized base pairs of probe and sample nucleic acids and causes the tertiary structure of the helix to unwind. An antibody specific for the newly formed antigenic determinant created by the intercalating agent and the unwound helix is detected by conventional means. This method lacks selectivity for the target hybrids because intercalating agents fail to recognize specific sequences. Furthermore, the antibodies recognize only the intercalating agent/nucleic acid complex, but do not detect a specific sequence. Therefore, additional selection or purification steps are required to prevent non-specific signal, making this time consuming and labor intensive approach poorly suited for clinical diagnosis.
Hybridization of the probe to the specific nucleic acid sequences may also be detected with the aid of an antibody specific for a labeled probe as described in U.S. Pat. No. 4,743,535 to Carrico. The probe is labeled with a detectable substance such as flavin adenine dinucleotide (FAD) or a fluorescent agent. An antibody specific for the labeled probe, after it has hybridized to the specific nucleic acid sequence, is detected by a biochemical reaction. This method of detection also creates non-specific binding and the likelihood of false-positive results and is not well suited for clinical screening.
Attempts have been made to increase the sensitivity of nucleic acid assays by target amplification. Methods of amplifying nucleic acid sequences are commercially available. These methods include the polymerase chain reaction (PCR), the ligation amplification reaction (LCR), and the transcription based amplification reaction (TMA). PCR technology is described in PCR Protocols A Guide to Methods and Applications by Michael A. Innis, David H. Gelfand, John J. Sninsky and Thomas J. White, pp. 39-45 and 337-385 (Academic Press, Inc., Harcourt Brace Jovanovich, Publishers, 1990). PCR technology is also described by Marx, J. L., Science 140:1408-1410 (1988) and in U.S. Pat. Nos. 4,683,195 and 4,683,202, to Mullis. Ligation amplification reaction is described by Wu, D. Y and Wallace, R. B, Genomics 4:560-569 (1989) and Barringer, K. J., et al., Gene 89:117-122 (1990). Transcription based amplification reaction is described by Kwoh, D. Y., et al., Proc. Natl. Acad. Sci. USA 86:1173-1177 (1989). These methods have the advantages of high sensitivity, but the disadvantages of having a lengthy, tedious, and expensive sample preparation, being prone to false-positive results from reaction product contamination, and having the inability to accurately quantify the initial amount of target nucleic acids. Amplification reaction products are most often detected by a hybridization assay.
The degree of sensitivity achieved in assays for the detection of nucleic acid molecules, either RNA or DNA, in a sample is generally lower for RNA than DNA because RNA is subject to degradation by endogenous RNAses in the sample, resulting in less RNA available for detection. In addition, background interference caused by contaminants in the sample is difficult to eliminate without causing further degradation of the target nucleic acid, such as RNA.
Hybridization assays for the detection of nucleic acid molecules, i.e. RNA, have been developed. For example, a hybridization protection assay for RNA is commercially available from Gen-Probe Inc. (San Diego, Calif.). The hybridization protection assay employs a single-stranded nucleic acid probe linked to an acridinium ester, as described by Engleberg, N. C., ASM News 57:183-186 (1991), Arnold et al. Clin. Chem. 35:1588-1594 (1989) and U.S. Pat. No. 4,851,330. Hybridization of the probe to a target RNA molecule protects the acridinium ester bond from heat hydrolysis so that the detected chemiluminescent signal is proportional to the amount of target RNA in the sample. The sensitivity of this protection assay is limited by background luminescence caused by non-hybridized probe.
Polyclonal and monoclonal antibodies and other similar entities are commonly used for detection purposes. Specifically, polyclonal antibodies recognize a plurality of epitopes, while monoclonal antibodies only recognize one specific epitope. Monoclonal antibodies which detect RNA:DNA hybrids are currently available. Polyclonal antibodies which detect RNA:DNA hybrids have been prepared, although, generally, they have not been as specific as the monoclonal antibodies, which are designed to bind to a specific epitope.
Monoclonal antibodies to RNA:DNA hybrids are now available. U.S. Pat. No. 4,732,847 to Stuart et al. and the publication of Stuart et al., Proc. Natl. Acad. Sci. USA 78:3751 (1981) describe a method of hybridization detection of specific nucleic acid sequences on a solid surface involving a monoclonal antibody specific for a poly(A)-poly(dT) duplex. In Stuart, annealing DNA or RNA sequences complementary to the sequence of interest forms RNA:DNA hybrids. Stuart specifically teaches against the use of polyclonal antibodies because with polyclonal antibodies, one cannot preclude significant binding to single- or double-stranded nucleic acids. Further, unlike the present invention described herein, Stuart does not contemplate the advantages of polyclonal antibodies for arrays of very short oligomers on glass or silicon chips. In addition, Stuart does not contemplate microarrays, especially high-density arrays on glass slides or silicon chips. Nor does Stuart disclose attaching a nucleic acid probe to the surface of a solid phase. Instead, Stuart fixes a sample polynucleotide to a surface, while probe (e.g., a predetermined nucleotide sequence) is present in the liquid phase. In view of the foregoing, the present invention provides significant benefits and advantages to the art.
Boguslawski et al., J. Immunol. Methods 89:123-130 (1986) developed a hybridization assay using anti-hybrid coated polystyrene beads isolated on filter paper in an attempt to reduce non-specific binding and avoid complicated washing procedures. A monoclonal antibody specific for RNA:DNA hybrids secreted by hybridoma HB 8730, is disclosed in U.S. Pat. No. 4,833,084 to Carrico et al. In Carrico, RNA:DNA hybrids formed by specific reannealing of a probe polynucleotide and the sequence of interest can be sensitively and specifically detected by binding to the monoclonal antibodies.
Microarrays refer to an orderly arrangement of distinct biological molecules, including RNA, DNA, protein, or the like, arrayed or immobilized to a solid substrate. These microarrays of binding agents, such as oligonucleotides and probes, have become an increasingly important tool in the biotechnology industry and related fields. Microarrays comprising a plurality of binding agents or elements are immobilized onto the surface of a solid support in an orderly fashion or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like. Elements as used herein in a microarray context, refer to hybridizable nucleic acid sequences, oligonucleotides, primers, probes, and/or amino acid sequences arranged in a distinct and identifiable manner on the surface of a substrate. Detection of biological molecules through the use of microarrays is beneficial for analyzing numerous samples and biological molecules, reducing the amount of sample required for analysis, decreasing experimental variability, decreasing sample preparation time, confirming results, and for decreasing costs of such analysis.
Currently, one of the primary uses of microarrays is to measure gene expression in biological samples. Gene expression measurements include detecting the presence or absence of mRNA or measuring increased or decreased concentrations of mRNA. In order to detect hybridization and to measure gene expression by conventional methods, however, the sample must first be purified and labeled. Two common techniques for purifying and labeling the sample are: 1) RNA amplification, labeling, and hybridization, and 2) cDNA labeling and hybridization. The amplification part of the first technique is described in U.S. Pat. Nos. 5,716,785 and 5,891,636 issued in 1998 and 1999, respectively, to Van Gelder et al. Highly purified total RNA or mRNA is used, which is an expensive and tedious time-consuming procedure. An oligo-dT primer is also used to reverse-transcribe the poly A-tailed mRNA into an anti-sense single-stranded cDNA. The oligo-dT further contains the sequence for T7 RNA polymerase on the 5 prime end of the dT sequences. After reverse transcription, a combination of RNAse H, DNA ligase, and DNA polymerase are used to generate a double stranded cDNA. Because the original RT primer contained a T7 RNA polymerase promoter, the double-stranded cDNA contains a full T7 RNA promoter. The double-stranded cDNA is then used as a template for T7 RNA polymerase. Approximately 100-1000 additional copies of RNA are generated from each copy of cDNA. During the transcription process, labeled nucleotides are incorporated into the transcribed RNA. Labeled RNA is then hybridized to the DNA microarray forming labeled RNA:DNA hybrids. Fluorescent labels may be detected directly while indirect labels may be detected after reaction with a secondary binding agent.
A second sample preparation technique produces and measures labeled cDNA. In this technique total RNA or mRNA is purified from the biological sample. An oligo-dT primer is used to reverse-transcribe the poly-A tailed mRNA into an anti-sense single-stranded cDNA. During the reverse-transcription, labeled nucleotides are incorporated into the nascent DNA strand. After synthesis, the RNA strand is destroyed. The labeled cDNA strand is then hybridized to the microarray. If the nucleotides were labeled with fluorescence, then the hybrids are visualized directly with a fluorescence array scanner. If the nucleotides were labeled with biotin, then the microarray is first reacted with labeled streptavidin and then scanned.
The disadvantages of both of these techniques are several fold. Firstly, both require a large quantity of highly purified nucleic acids (i.e. RNA or DNA). Purification requires additional steps which are time consuming and labor intensive. In addition, these techniques are inaccurate. Reverse transcription occurs at different efficiencies and kinetic rates depending on the nucleic acid sequences, artificially changing the concentration of specific nucleic acid sequences. Prokaryotic mRNA and some eukaryotic mRNA do not contain the poly A sequence or tail at the 3 prime end or the poly A tail may be degraded during purification, and therefore cannot be labeled or detected with the current techniques since there is no sequence to prime the reverse transcriptase step. The current techniques are thus restrictive to the types of samples which can be used for detection. Also, these methodologies involve labeled nucleotides. The incorporation of labeled nucleotides into unlabeled nucleic acids occurs at a lower efficiency and at a slower rate than natural nucleotides. Once more, labels may be incorporated with different efficiencies depending on the sequence. Therefore, the label density may differ between different sequences, artificially changing the measured amount of these nucleic acids. Thus, quantification is only relative. Labeled nucleic acids also exhibit different hybridization kinetics than natural nucleic acids, usually rendering them less specific. In addition, the present methods may require higher stringency hybridization conditions than unmodified nucleotides to achieve the same level of specificity. However, use of the higher stringency conditions to achieve acceptable specificity will lower the sensitivity of detection. Consequently, there is a need for an assay for detection and for quantitative analysis of biological molecules, including DNA, RNA, protein, and the like, that is accurate, both time and cost efficient, and capable of screening one or more sample biological molecules with great sensitivity and minimal non-specific binding.
Therefore, it may be useful to have a method to detect and measure the amount of one or more biological molecules, including, but not limited to RNA, DNA, or protein, that is easy to use, highly specific, accurate, and sensitive for screening biological molecules.
Accordingly, it is an object of the invention to provide an assay to detect the absence or presence, and quantify biological molecules, including, but not limited to RNA, DNA, or protein.
It is also an object of the present invention to provide a method of detecting an RNA:DNA hybrid comprising a specific target first biological molecule in a sample and a second biological probe.
It is an object of the present invention to provide a sensitive and quantitative assay having minimal false positives.
It is a further object of the present invention to provide an assay for massive parallel screening.
Disclosed is an assay for detecting and measuring a biological molecule of interest, including RNA, DNA, protein, and the like, in a sample by hybridizing the biomolecule to a complementary biomolecule probe forming double-stranded hybrids, followed by immunological detection of these double-stranded hybrids formed on a solid phase with an antibody or other entity which specifically recognizes RNA:DNA hybrids and is detectable. This method may be used to detect the presence of one or more specific biological molecules present in a variety of samples.
This invention provides for a method of simultaneously monitoring the amount (e.g. detecting and quantifying the amount) of a multiplicity of biological molecules.
The present invention relates to an assay for detecting RNA:DNA hybrids using detectably labeled entities specific for recognizing RNA:DNA hybrids. Preferably, the entity is a detectably labeled RNA:DNA hybrid-specific antibody or a fragment thereof. The antibody used for detecting the RNA:DNA hybrids may be monoclonal or polyclonal, and preferably polyclonal for detection of short biological molecule probes having a length of less than 30 bases.
The present invention also relates to an assay using the microarrays of the invention to determine physiological responses by gene expression, polymorphism mutation detection, SNP analysis, or the like. The method may be used to detect any and all genotypic variations, including insertion or deletion mutations.
Further, the present invention relates to an assay utilizing reverse transcriptase for extending short biological molecules, thereby enhancing detection of RNA:DNA hybrids. Preferably, the reverse transcriptase is thermostable and lacks RNAse H function.
The present invention further relates to a kit for the detection and quantification of biological molecules, wherein the kit may be used to screen samples for large numbers of targets described herein by the present invention.